Production of recombinant HPV11/16 E6/E7-MBP-His6 fusion proteins and their potential to induce cytokine secretion by immune cells in peripheral blood

Human papillomavirus (HPV) infection poses a significant threat to public health worldwide. Targeting the function of HPV E6 and E7 proteins and activating the host immune response against these proteins represent promising therapeutic strategies for combating HPV-related diseases. Consequently, the efficient production of soluble, high-purity E6 and E7 proteins is crucial for function and host immune response studies. In this context, we selected the pMCSG19 protein expression vector for Escherichia coli to produce soluble MBP-His6 tagged HPV11/16 E6/E7 proteins, achieving relatively high purity and yield. Notably, these proteins exhibited low toxicity to peripheral blood mononuclear cells (PBMCs) and did not compromise their viability. Additionally, the recombinant proteins were capable of inducing the secretion of multiple cytokines by immune cells in peripheral blood, indicating their potential to elicit immune responses. In conclusion, our study offers a novel approach for the production of HPV11/16 E6/E7 fusion proteins with relatively high purity and yield. The fusing HPV11/16 E6/E7 proteins to MBP-His6 tag may serve as a valuable method for large-scale protein production in future research endeavors.


Introduction
Human papillomavirus (HPV)infection is one of the most prevalent sexually transmitted diseases worldwide.Low-risk HPV types, particularly HPV6 and HPV11, are associated with genital warts, recurrent respiratory papillomatosis, and oral papillomas.Persistent infection with high-risk HPV types, such as HPV16 and HPV18, contributes to cervical, oropharyngeal, and cutaneous cancers [1].The consistent and stable expression of E6 and E7 oncoproteins in HPV-positive cells is crucial for persistent HPV infection and the development of malignant phenotype [2,3].These oncoproteins have been the focus of numerous studies aimed at understanding the molecular mechanisms of HPV-induced carcinogenesis and the development of therapeutic vaccines and diagnostic tools [4,5].In this respect, the efficient production of soluble, high-purity E6 and E7 proteins is essential for advancing our understanding of HPV-associated diseases and fostering the development of innovative medical interventions to combat these illnesses.
Although eukaryotic expression systems, such as insect or mammalian cells, allow for proper protein folding and post-translational modifications, these systems are expensive, time-consuming, and lower yield [6]. Bacterial systems, such as Escherichia coli, offer several advantages for recombinant protein expression, including rapid growth, high yield, simplicity, ease of use, costeffectiveness, and high-throughput capabilities.However, the production of soluble and pure HPV E6 proteins in bacteria poses significant challenges due to the protein's structure.The main focus has been on the HPV16 type when producing E6 proteins in bacteria [7][8][9][10].This protein possesses numerous cysteine residues, encouraging the formation of disulfide bonds [9].The extensive formation of these bonds restricts the exposure of hydrophilic residues to the solvent, thereby reducing protein solubility and causing protein misfolding.This, in turn, leads to protein aggregation and the creation of insoluble protein complexes.Consequently, aggregates form during overexpression in cells and persistently increase during purification [8].Similarly, when E7 protein is overexpressed in bacteria, it mainly exists as insoluble inclusion bodies due to overloading the host protein folding machinery [11,12].However, unlike the cysteine-rich E6 protein that is susceptible to forming disulfide bond bridges, the E7 protein's structure exhibits enhanced solubility [13].Generally, the overexpression of heterologous proteins in a host necessitates strategies to improve their solubility and facilitate their correct folding.
Fusion tags, such as glutathione S-transferase (GST) or maltose-binding protein (MBP), can improve the solubility of fusion proteins.In this strategy, the MBP tag, when fused to the N-terminus of the target protein, can significantly enhance its solubility, preventing aggregation and facilitating proper folding [14,15].This is particularly beneficial for proteins that are prone to forming inclusion bodies or are difficult to express in a soluble form.Although MBP could increase the solubility of HPV16 E6 protein, some proteins still inevitably aggregate [16,17].Moreover, the hydrophobic nature of E6 proteins can lead to protein aggregation and loss during chromatography steps [15,18].
One way around this obstacle is to fuse the desired protein to a dual-affinity tag designed in tandem with a protease cleavage site.The combination of the MBP and hexahistidine (His 6 ) tags provides complementary benefits in recombinant proteins [19].Fusing MBP to the N-terminus of target proteins increases their solubility and expression levels in the host system, facilitating their purification and analysis in their native and functional state.The His 6 tag enables easy purification of the recombinant protein using immobilized metal affinity chromatography (IMAC).The strong and specific interaction between the His 6 tag and metal ions (e.g., Ni 2+ or Co 2+ ) allows for a rapid and selective purification process, resulting in a high degree of purity.
In this study, we inserted the E6 and E7 segments of HPV11 and HPV16 into the plasmid pMCSG19, a bacterial vector with an MBP-TVMV-6xHis-TEV leader, to obtain E6/E7 proteins with an MBP-His 6 tag.We then expressed and purified the recombinant HPV11/16 E6/ E7 proteins with a fused MBP-His 6 tag in a prokaryotic expression system.Finally, we assessed their impact on the cytotoxicity, cell viability, and cytokine secretion by immune cells in peripheral blood.

Materials and methods
Expression of HPV11 E6-MBP-His 6 , HPV11 E7-MBP-His 6 , HPV16 E6-MBP-His 6 , and HPV16 E7-MBP-His 6 proteins in Escherichia coli cells The E6 and E7 gene sequences of HPV11 (low-risk) and 16 (high-risk) (Table 1) were obtained from the NCBI Nucleotide database (https:// www.ncbi.nlm.nih.gov/ nucle otide).The plasmid pMCSG19, a bacterial vector with an MBP-TVMV-6xHis-TEV leader, was chosen as the vector to express the E6 and E7 proteins.Homologous sequences complementary to the plasmid vector insertion site were added to the 5' ends of the forward primers and reverse primers of HPV11/16 E6/E7 genes (Table 2).The primers were designed and validated using the NCBI Primer-BLAST server (https:// www.ncbi.nlm.nih.gov/ tools/ primer-blast/).Initially, the primers with homologous sequences were employed to amplify the HPV11/16 E6/E7 genes via PCR.Next, the target products were purified using a Gel DNA Recovery Kit (Bioteke Corporation, Beijing, China) subjected to an 'In Fusion' reaction with the pMCSG19 plasmid (Huayueyang Biotechnology Ltd., Beijing, China).In this process, the homologous sequences of the target gene and the plasmid vector combined to form a recombinant plasmid.Subsequently, the PCR reaction was treated with the DpnI restriction enzyme and introduced into DH5α competent Escherichia coli using the heat shock method, as previously described [20].Finally, the constructed plasmids were extracted from E. coli and confirmed by sequencing (Sangon Biotech, Shanghai, China).
Target plasmids were transformed into BL21(DE3) competent E. coli cells for protein expression, and the empty plasmid pMCSG19 was transformed as a control group.The cells were cultivated in an LB medium containing 100 µg/mL ampicillin in a shaking incubator at 37 °C.When the optical density of the medium reached 0.6 at 600 nm, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM to induce protein expression.After 5 h of induction, the

Purification of the MBP-His 6 tagged HPV11/16 E6/E7 proteins
For protein purification, the cells were re-suspended in PBS and disrupted by sonication.The supernatants were applied to an equilibrated Ni-nitrilotriacetic acid (NTA) resin column (Sangon Biotech, Shanghai, China), a nickel-charged affinity resin for affinity purification of His-tagged fusion proteins.Then, we performed a stepwise elution using different concentrations of imidazole (20 mM, 50 mM, and 300 mM) to optimize the elution conditions for both the MBP-His 6 tag and the HPV11/16 E6/E7-MBP-His 6 fusion proteins.The optimal imidazole concentration for eluting the MBP-His 6 tag was 50 mM, while the HPV11/16 E6/E7-MBP-His 6 fusion proteins were best eluted with 300 mM imidazole.These specific concentrations were chosen because they provided the best balance between protein purity and yield, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The potential endotoxin in the elution fraction was removed by flowing through polymyxin B-agarose (Sigma-Aldrich, St. Louis, Missouri, USA) and then detected by Chromogenic LAL Endotoxin Assay Kit (GenScript, Nanjing, China) following the manufacturer's instructions.Next, the buffer of purified proteins was exchanged with sterile PBS and concentrated using Amicon Ultra centrifugal filters.Finally, the concentrations of the purified proteins were determined using a Pierce bicinchoninic acid (BCA) protein assay kit (Invitrogen, Carlsbad, CA, USA).The expression and purity of the proteins were validated by SDS-PAGE followed by Coomassie Brilliant Blue Staining as described in a previous study [21].The recombinant proteins were stored in liquid nitrogen.

Isolation and culture of peripheral blood mononuclear cells
Human peripheral blood mononuclear cells (PBMCs) were isolated from blood samples using the Isopaque-Ficoll method (TBD, Tianjin, China) according to the manufacturer's instructions.Blood samples were collected from healthy volunteers, and informed consent was obtained from all subjects involved in the study.PBMCs were cultured at 1 × 10 6 cells/mL density in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% Fetal Bovine Serum (FBS; Invitrogen, Carlsbad, CA, USA) in a humidified incubator at 37 °C with 5% CO 2 .

Stimulation of PBMCs with the recombinant HPV11/16 E6/ E7-MBP-His 6 proteins
PBMCs were isolated on the day of the experiment and plated on a 96-well plate at a density of 1 × 10 5 cells/ well.The recombinant proteins of HPV11 E6-MBP-His 6 , HPV11 E7-MBP-His 6 , HPV16 E6-MBP-His 6 , and HPV16 E7-MBP-His 6 were diluted with sterilized PBS and added to the wells at the final concentrations of 1, 10, and 100 µg/mL, respectively.MBP-His 6 protein treatments at corresponding concentrations were performed as controls.Then, the supernatants were collected at 24 h poststimulation for further detection of secreted cytokines.

Challenging mice with recombinant HPV11/16 E6/ E7-MBP-His 6 proteins
The animal experiment was conducted according to the Animal Care and Use Procedure (ACUP) guidelines.Specific pathogen-free (SPF) BALB/c mice (6-8 weeks old) were challenged by the tail vein injection with the recombinant proteins.The mice were housed in cages under SPF conditions with a natural light-dark cycle, a temperature of 21 °C, relative humidity between 40 and 60%, and food and water ad libitum in the Experimental Animal Center of Nanfang Hospital, Southern Medical University.Mice were administered a single intravenous injection of the recombinant HPV11 E6-MBP-His 6 , HPV11 E7-MBP-His 6 , HPV16 E6-MBP-His 6 , HPV16 E7-MBP-His 6 , and MBP-His 6 proteins, with each mouse receiving 200 µg of protein.The experiment was conducted three times, with at least five mice per group in each repetition.Blood samples were collected from the orbital venous plexus of the mice 24 h post-injection, and serum was isolated from the blood for the detection of secreted cytokines.
Table 2 Primers for inserting the E6 or E7 segments into plasmid pMCSG19 using 'In-fusion' PCR The underlined sections represented homologous sequences complementary to the plasmid vector insertion site, which were added to the 5′ ends of the forward primers and reverse primers of the target genes.

Evaluation of the impact of recombinant proteins on cytotoxicity of PBMCs
The cytotoxicity of recombinant HPV11/16 E6/E7-MBP-His 6 proteins was determined using lactate dehydrogenase (LDH) assay kits (Beyotime, Shanghai, China).In detail, the cell supernatants were collected after 24 h of the stimulation with recombinant proteins and then subjected to the detection of LDH activity according to the manufacturer's instructions.Cytotoxicity (%) = [(Protein-treated LDH activity -PBS-treated LDH activity) / (Maximum LDH release activity -PBS-treated LDH activity)] × 100.

Evaluation of the impact of recombinant proteins on PBMC viability
The PBMC viability was measured by the CCK-8 kit (Fude Biological Technology, Hangzhou, China).Briefly, 10 µL CCK-8 solution was added to each well for a further 3 h at 37 °C.The optical density (OD) values of the reactant were measured at 450 nm wavelength using a spectrophotometer (ELX800, BioTek Instruments, Winooski, USA).Six replicated wells were carried out for each experiment.The cell viability was calculated by the following: cell viability (%) = [(OD experiment -OD blank) / (OD control -OD blank)] × 100.The experiments were repeated in triplicate with three replicates each.

Evaluation of the impact of recombinant proteins on cytokine secretion of PBMCs and mouse serum
The concentration of 12 cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17 A, IL-17 F, and IL-22) in the culture supernatants of PBMCs and mouse serum was quantified by LEGENDplex Human Th cytokine panel (BioLegend, San Diego, CA, USA) on the flow cytometer (BD LSRFortessa, Franklin Lakes, USA) under close compliance with the manufacturer's guidelines.The experiments were repeated in triplicate with three replicates each.

Statistical analysis
Each experiment was repeated in triplicate.Data were analyzed using the SPSS software 15.0 (SPSS Inc., Chicago, IL, USA).The analytes for cytokine levels of each group were compared using a one-way ANOVA test with Bonferroni's correction.The results were expressed as the means ± standard errors of the means (SEM), and the differences were considered significant when p < 0.05.

Results
Recombinant HPV11/16 E6/E7-MBP-His 6 proteins were produced in a prokaryotic expression system HPV11/16 E6/E7 was inserted into the C-terminus of the MBP-His 6 tag in the E. coli plasmid pMCSG19 by In-fusion PCR.Primers with homologous sequences complementary to the plasmid vector insertion site were used to amplify the HPV11/16 E6/E7 genes via PCR.Electrophoresis result revealed that the PCR product sizes corresponded to the expected sizes of HPV11 E6 (493 bp), HPV11 E7 (337 bp), HPV16 E6 (517 bp), and HPV16 E7 (337 bp) (Fig. 1a).Nucleotide sequence analysis confirmed the accuracy of target sequences from the recombinant vectors (Fig. 1b).Target proteins were detected in the total lysates of E. coli cells by SDS-PAGE.
The protein bands of MBP-His 6 , HPV11 E6-MBP-His 6 , HPV11 E7-MBP-His 6 , HPV16 E6-MBP-His 6 , and HPV16 E7-MBP-His 6 proteins were located approximately at 43 kDa, 61 kDa, 54 kDa, 62 kDa, and 54 kDa regions, respectively (Fig. 1c, left).The thick protein bands suggested their abundant production and low cytotoxicity to the host cells.Then, the target proteins were purified.Endotoxin concentrations of the purified proteins were below 0.1 EU/mL.High purity of production was validated by SDS-PAGE analysis, presenting as a major band with the expected molecular weight in each lane (Fig. 1c, right).The thin bands of 11/16 E6-MBP-His 6 proteins in SDS-PAGE implied relatively poor solubility, possibly due to inclusion bodies formation [17].The procedure described above allowed a 4-6 mg yield in the case of 11/16 E6-MBP-His 6 proteins and 24-30 mg in the case of 11/16 E7-MBP-His 6 proteins from 1 L of cultured cells.Surprisingly, the yield of 11/16 E7-MBP-His 6 proteins was close to that of MBP-His 6 .Still, the purified proteins were adequate for subsequent experiments.We attempted to remove the MBP-His 6 tag, but the solubility of the target proteins significantly decreased, especially for the E6 proteins, resulting in a failure to obtain sufficient amounts of E6 and E7 proteins.This illustrates the importance of the MBP-His 6 tag in maintaining the solubility of E6 and E7.In the subsequent experiments, we used the MBP-His 6 tag as a control to clarify the cytotoxicity and immunogenicity of E6/E7 proteins in the fusion proteins.

Recombinant HPV11/16 E6/E7 proteins had low toxicity to PBMCs and did not affect their viability
LDH release assay was performed to evaluate the cytotoxicity of HPV11/16 E6 and E7 proteins on PBMCs.As shown in Fig. 2a, 24 h after stimulating PBMCs with the recombinant proteins, LDH release slightly increased in a dose-dependent manner in all groups.In this context, all the treatments with recombinant proteins from 1 to 100 µg/mL resulted in approximate 26-30% increases in LDH activity.However, there was no significant difference in the LDH activity between HPV11/16 E6/ E7-MBP-His 6 -treated groups and the MBP-His 6 -treated group.These results indicated that HPV11/16 E6/ E7-MBP-His 6 proteins had low toxicity to the peripheral immune cells.Next, we detected the viability of PBMCs at 24 h after treatment with the recombinant proteins (1-100 µg/ mL).Compared to the MBP-His 6 treatment, HPV11/16 E6/E7-MBP-His 6 treatments exhibited no significant impact on cell viability (Fig. 2b).Moreover, as the protein concentrations increased, there was also no significant change in cell viability.Thus, we suggested that the recombinant HPV11/16 E6 and E7 proteins had little effect on the viability of PBMCs.

Discussion
In this study, we successfully fused HPV11 E6, HPV11 E7, HPV16 E6, and HPV16 E7 proteins to the MBP-His 6 tag and obtained the target proteins with relatively high purity and yield.These proteins exhibited low toxicity Fig. 1 Recombinant and purification of HPV11/16 E6/E7-MBP-His 6 proteins.a PCR products of HPV11 E6, HPV11 E7, HPV16 E6, and HPV16 E7 were visualized using gel electrophoresis.b Nucleotide sequence analysis for target sequences from the recombinant vectors.c The target protein bands were detected in the total lysates of E. coli cells (left) and purified products (right) using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie Brilliant Blue Staining to PBMCs and did not compromise their viability.Furthermore, they were capable of inducing the secretion of multiple cytokines by immune cells in peripheral blood, suggesting their bioactive potential in eliciting immune responses.
Recombinant protein expression technology is an essential tool for studying protein function, structure, vaccine synthesis, and screening targeted drugs.In the process of generating recombinant proteins, the N-terminal or C-terminal of the target protein is often fused and expressed with other specific proteins, peptides, or oligopeptide tags.This practice can not only retain the structure of natural proteins, but also increase solubility, prevent degradation, promote secretion, and facilitate purification [23].It has been reported that MBP significantly improves solubility when fused to the N-termini of various target proteins, but this effect is considerably less pronounced when attached to their C-termini [14,24,25].Besides, the His 6 tag enables easy and efficient purification of the target protein using IMAC.In our study, the MBP-His 6 was fused to the N-termini of E6 and E7 proteins.The dual-affinity tag system MBP-His 6 enabled us to obtain soluble and pure proteins, which is essential for downstream functional and structural analyses.Specifically, the yield of MBP-His 6 -tagged E6 protein ranges from 4 to 6 mg per liter of cultured cells, whereas the yield of MBP-His 6 -tagged E7 protein reaches a higher level of 24 to 30 mg per liter of cultured cells.The yields of HPV11/16 E6-MBP-His 6 proteins obtained by this method could fully meet general experimental needs.
After protein purification using IMAC, the concentration of soluble E7-MBP-His 6 proteins remained abundant, while the concentration of soluble E6-MBP-His 6 proteins was significantly reduced.This phenomenon may be attributed to the poor solubility and aggregation properties of E6 proteins in a recombinant way.When the E6 protein was efficiently expressed in prokaryotic cells, it tends to misfold and form inclusion bodies [26].Some researchers have used a prokaryotic expression vector to fuse MBP with HPV16 E6 protein to improve solubility.Although they found that the solubility of E6 protein was enhanced, some proteins still inevitably aggregated [15,27].Inclusion body formation depends on the rate of protein folding and aggregation.Lowering the growth temperature of recombinant bacteria is the most common method to reduce inclusion body formation, but it is time-consuming [28].We attempted to grow E. coli at room temperature for HPV11/16 E6-MBP-His 6 expression but failed to significantly increase their solubility.Further research is needs to explore approaches to improve the solubility of E6 proteins.Additionally, we tried to remove the MBP-His 6 tag and observed a dramatic decrease in the solubility of the target protein, particularly the E6 proteins.As a result, we were unable to obtain sufficient amount of proteins for our experiments.The highly soluble MBP plays a crucial role in preventing the precipitation of particles produced by E6.However, once the E6 protein is partially dissociated from MBP through protease hydrolysis, it precipitates instantly [8].
Considering that E6 and E7 proteins are critical targets in immunotherapy for HPV-related diseases, we measured the biological activity of E6/E7 proteins in the MBP-His 6 -fused HPV11/16 E6/E7 proteins, including their cytotoxicity and impacts on the cell viability and cytokine secretion by immune cells in peripheral blood.Consistent with the computational analysis of a previous study, E6 and E7 proteins exhibited low toxicity to the host immune system [29].Interestingly, we observed a significantly increase in the secretion of multiple cytokines from PBMCs and mice serum after challenging with recombinant HPV11/16 E6/E7-MBP-His 6 proteins.Notably, these cytokines are involved in both innate immune response and adaptive immune response.In this study, cytokine profiles in the in vivo and in vitro experiments were evaluated after 24 h, indicating an early immune response but not dynamic changes.However, the overall results support the potential biological activity of recombinant HPV11/16 E6/E7-MBP-His 6 proteins.
Recent research has indicated that the MBP-His 6 or MBP tag can enhance the immunogenicity of target protein [30][31][32].This finding holds significant implications for the development of therapeutic vaccines using the MBP-His 6 tag.The enhancement of immunogenicity by the MBP-His 6 tag is primarily attributed to the properties of the MBP component.One contributing factor is the relatively large molecular weight of MBP, approximately 42 kDa.When fused with the target protein, MBP increases the molecular weight of the entire fusion protein, thereby improving its recognition by the immune system.Additionally, MBP, being an exogenous protein derived from E. coli, may be perceived as a non-self protein in mammals, which could trigger an immune response, behaving like an adjuvant.Furthermore, the stable structure of MBP aids in the correct folding of the target protein.Correctly folded proteins are more likely to be recognized as antigens by the immune system, thereby eliciting an immune response [33].Lastly, MBP has the ability to enhance the solubility of the target protein, increasing its bioavailability within the body.Proteins with greater solubility are more readily recognized and processed by the immune system.Therefore, studying the impact of the MBP-His 6 tag on the immunogenicity of the target protein may provide a promising direction for the development of HPV therapeutic vaccines.However, our study was limited by the unavailability of sufficient HPV11/16 E6/E7 proteins as a control for MBP-His 6 -tagged HPV11/16 E6/E7 proteins.

Conclusions
Taken together, our research presents a new method for the production of MBP-His 6 -tagged HPV11/16 E6/ E7 proteins with relatively high purity and yields, which possess the potential to induce cytokine secretion.The fusion of MBP-His 6 tag to the HPV11/16 E6/E7 proteins may be used for large-scale protein production in the future.Future studies should focus on the effect of MBP-His 6 tag on the immunogenicity of E6 and E7 proteins.
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FAAC 29 HPV11 28 HPV16
E6 ATG GAA AGT AAA GAT GCC TCC ACG TCT GCA ACA TCT ATA GAC CAG TTG TGC AAG ACG TTT AAT CTT TCT TTG CAC ACT CTG CAA ATT CAG TGC GTG TTT TGC AGG AAT GCA CTG ACC ACC GCA GAG ATA TAT GCA TAT GCC TAT AAG AAC CTA AAG GTT GTG TGG CGA GAC AAC TTT CCC TTT GCA GCG TGT GCC TGT TGC TTA GAA CTG CAA GGG AAA ATT AAC CAA TAT AGA CAC TTT AAT TAT GCT GCA TAT GCA CCT ACA GTA GAA GAA GAA ACC AAT GAA GAT ATT TTA AAA GTG TTA ATT CGT TGT TAC CTG TGT CAC AAG CCG TTG TGT GAA ATA GAA AAA CTA AAG CAC ATA TTG GGA AAG GCA CGC TTC ATA AAA CTA AAT AAC CAG TGG AAG GGT CGT TGC TTA CAC TGC TGG ACA ACA TGC ATG GAA GAC TTG TTA CCC TAA MESKDASTSATSIDQLCKTFNLSLHTLQIQCVFCRNALTTAEIYAYAYKNLKVVWRDNFP-E7 ATG CAT GGA AGA CTT GTT ACC CTA AAG GAT ATA GTA CTA GAC CTG CAG CCT CCT GAC CCT GTA GGG TTA CAT TGC TAT GAG CAA TTA GAA GAC AGC TCA GAA GAT GAG GTG GAC AAG GTG GAC AAA CAA GAC GCA CAA CCT TTA ACA CAA CAT TAC CAA ATA CTG ACC TGT TGC TGT GGA TGT GAC AGC AAC GTC CGA CTG GTT GTG GAG TGC ACA GAC GGA GAC ATC AGA CAA CTA CAA GAC CTT TTG CTG GGC ACA CTA AAT ATT GTG TGT CCC ATC TGC GCA CCA AAA CCA TAA MHGRLVTLKDIVLDLQPPDPVGLHCYEQLEDSSEDEVDKVDKQDAQPLTQHYQILTCC CGC DSNVRLVVECTDGDIRQLQDLLLGTLNIVCPICAPKP 10889.46HPV16 E6 ATG CAC CAA AAG AGA ACT GCA ATG TTT CAG GAC CCA CAG GAG CGA CCC AGA AAG TTA CCA CAG TTA TGC ACA GAG CTG CAA ACA ACT ATA CAT GAT ATA ATA TTA GAA TGT GTG TAC TGC AAG CAA CAG TTA CTG CGA CGT GAG GTA TAT GAC TTT GCT TTT CGG GAT TTA TGC ATA GTA TAT AGA GAT GGG AAT CCA TAT GCT GTA TGT GAT AAA TGT TTA AAG TTT TAT TCT AAA ATT AGT GAG TAT AGA CAT TAT TGT TAT AGT TTG TAT GGA ACA ACA TTA GAA CAG CAA TAC AAC AAA CCG TTG TGT GAT TTG TTA ATT AGG TGT ATT AAC TGT CAA AAG CCA CTG TGT CCT GAA GAA AAG CAA AGA CAT CTG GAC AAA AAG CAA AGA TTC CAT AAT ATA AGG GGT CGG TGG ACC GGT CGA TGT ATG TCT TGT TGC AGA TCA TCA AGA ACA CGT AGA GAA ACC CAG CTG TAA E7 ATG CAT GGA GAT ACA CCT ACA TTG CAT GAA TAT ATG TTA GAT TTG CAA CCA GAG ACA ACT GAT CTC TAC TGT TAT GAG CAA TTA AAT GAC AGC TCA GAG GAG GAG GAT GAA ATA GAT GGT CCA GCT GGA CAA GCA GAA CCG GAC AGA GCC CAT TAC AAT ATT GTA ACC TTT TGT TGC AAG TGT GAC TCT ACG CTT CGG TTG TGC GTA CAA AGC ACA CAC GTA GAC ATT CGT ACT TTG GAA GAC CTG TTA ATG GGC ACA CTA GGA ATT GTG TGC CCC ATC TGT TCT CAG AAA CCA TAA MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCK-CDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP 11022.32 cells were harvested by centrifugation and washed with cold PBS.

Fig. 2
Fig. 2 The impact of recombinant HPV11/16 E6/E7-MBP-His 6 proteins on the cytotoxicity and cell viability of PBMCs.a The cytotoxicity of the recombinant proteins on PBMCs was evaluated using an LDH release assay.b The viability of PBMCs after the recombinant proteins treatment was detected by CCK-8 assay.Treatment with the MBP-His 6 protein was used as the control.The experiments were done in triplicate, and data from representative ones were demonstrated